Tubulointerstitial Research Articles

Fibrotic Mechanisms in Idiopathic Rapidly Progressive Glomerulonephritis: The Role of TGF-β1 and C5b-9

Background. Idiopathic IRPGN is a form of renal vasculitis in which a high chronicity index is present despite minimal impairment of renal function. The present study investigated the mechanisms underlining the relatively early appearance of fibrosis. Methods. In all, 34 patients (17 males) with biopsy proven idiopathic RPGN were included. On light microscopy, the percentage and evolution stage of crescents, the presence of glomerular necrosis, the degree or severity of arteriosclerosis, as well as the extent of tubulointerstitial (TIN) infiltration, interstial fibrosis, and tubular atrophy were assessed. Monoclonal antibodies were used to identify infiltrating macrophages, HLA-DR (+), α-SMA (+), and PCNA (+) cells, the expression of the adhesion molecule ICAM-1, the growth factor TGF-β1, and the terminal complement component C5b-9. Results. The presence of glomerular necrosis correlated positively with the number of SMA (+) cells in TIN (p = 0.036). Glomerular TGF-β1 expression had positive correlation with tubular C5b-9 expression. The tubulointerstitial TGF-β1 expression correlated with tubular C5b-9 expression (p = 0.001) and TGF-β1 expression (p = 0.009). Independent factors predicting the severity of renal function impairment were the CRP levels (p = 0.002) and the degree of arteriosclerosis (p = 0.01). CRP levels correlated with the severity of interstitial infiltration and fibrosis (p = 0.02), the expression of TGF-β1 in the glomeruli (p = 0.009) and the interstitial space (p = 0.001), and the intensity of tubular ICAM-1 and C5b-9 expression (p = 0.023, p = 0.002, respectively). The severity of proteinuria showed a significant correlation with the expression of TGF-β1 in the glomeruli (p = 0.033) and the tubulointerstitium (p = 0.019). Conclusions. The activation of interstitial fibroblasts seems to be an early phenomenon that is related to the extent of glomerular necrosis. Glomerular TGF-β1 may induce tubular expression of C5b-9. Increased tubular C5b-9 expression may result in interstitial fibrosis through increased TGF-β1 production.

Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1

To illuminate the possible role of Prohibitin (PHB) in tubulointerstitial fibrosis. (1) Forty-eight renal biopsy specimens were obtained from the patients with various primary glomerulonephritis, 26 male and 22 female, aged 7.5 +/- 5.5 (2.5 - 13 years), and nine kidney tissue specimens were obtained form the tissues far away from the tumor tissues and confirmed by pathological examination as normal tissues in the kidney dissected during operation as normal control. Immunohistochemistry was used to detect the protein expression of PHB and alpha-smooth muscle actin (alpha-SMA). The correlation between PHB and degree of tubulointerstitial lesion was compared. (2) Rat kidney fibroblastoma cells of the line NRK-49F were cultured, and laser scanning confocal microscopy was used to observe the subcellular location of PHB protein. The changes of PHB protein and mRNA expression in the NRK-49F cells upon TGF-beta1 stimulation were detected by Western blotting and RT-PCR analysis. (3) PHB expression plasmid was constructed and transfected into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to detect the PHB and alpha-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-beta1. (1) PHB protein expression was found in the normal renal tissues by immunohistochemistry, with a positive distribution in the interstitial cells and tubular epithelial cells. PHB was strongly down-regulated in the damaged interstitial and tubular epithelial cells, the higher the grade of damage, the lower the expression of PHB (all P < 0.01), and the PHB expression amount was negatively correlated with the degree of tubulointerstitial lesions (r = -0.802, P < 0.01). (2) Confocal microscopy showed that PHB was mainly located in the cytoplasm and weakly expressed in the nucleus of the NRK-49F cells. Treated with TGF-beta1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both time-dependently and dose-dependently (all P < 0.01). (3) A recombinant pcDNA3.1 (-)/PHB plasmid was successfully constructed. PHB protein expression in the transfected NRK-49F cells was 2.54 times higher compared with the non-transfected cells. (4) The proportions of the cells in the S and G(2)/M phases were higher in the NRK-49F cells stimulated by TGF-beta1, however, more NRK-49F cells remained in the G(0)/G(1) phase after transfection of PHB (P < 0.01). (5) Both alpha-SMA protein and mRNA were not expressed in the control cells while de novo expression of alpha-SMA in the NRK-49F cells was increased after the treatment of TGF-beta1. Over-expression of PHB did not affect he basic alpha-SMA expression but dramatically repressed TGF-beta1-initiated alpha-SMA expression in the NRK-49F cells (P < 0.01). PHB protein is expressed in the normal renal tissues and adversely correlated with the degree of tubulointerstitial lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-beta1.

Heme-Oxygenase Upregulation Ameliorates Angiotensin II-Induced Tubulointerstitial Injury and Salt-Sensitive Hypertension

Background: Heme oxygenase-1 (HO-1) has been implicated in the modulation of several diseases including hypertension (HTN) and renal injury. The tubulointerstitial (TI) injuries are supposed to be the main determinants for the development of salt-sensitive HTN. Therefore, this study examined the role of HO-1 in angiotensin II (AngII)-induced TI injury and salt-sensitive HTN. Methods: Sprague-Dawley rats on a high salt diet were treated by AngII infusion plus either hemin, an inducer of HO-1, or hemin + zinc protoporphyrin, a HO-1 inhibitor, for 2 weeks, and then followed for 6 weeks. Results: The AngII infusion resulted in acute HTN and proteinuria. Light microscopy revealed focal areas of tubular atrophy with mononuclear cell infiltration and interstitial expansion. The overexpression of osteopontin and TGF-β₁ accompanied by diminished expression of rat endothelial cell antigen-1, the hallmarks of TI injury, were observed. At 2 weeks, all interventions were withdrawn and systolic blood pressure returned towards normal. After a brief normal salt diet, rats were again placed on high salt diet, resulting in progressive increase in systolic blood pressure in the HO-1-inhibited group. Conclusion: The induction of HO-1 attenuated the development of HTN, suggesting that HO-1 plays a crucial role in significant attenuation of AngII-mediated TI injury and resultant salt-sensitive HTN.

Urinary Excretion of Monocyte Chemoattractant Protein-1: A Biomarker of Active Tubulointerstitial Damage in Patients with Glomerulopathies

Background/Aims: The urinary concentration of the monocyte chemoattractant protein-1 (uMCP-1) chemokine is increased in several proteinuric and/or inflammatory renal diseases. In the present study, we evaluated the association between uMCP-1 and renal function, proteinuria, glomerular and interstitial macrophage infiltration, and renal fibrosis in patients with primary and secondary glomerulopathies diagnosed by renal biopsy. Methods: Thirty-seven patients aged 32.6 ± 7.7 years were studied. uMCP-1 was determined by ELISA. Renal macrophage expression (CD68 positive cells) is reported as number of macrophages/10⁴ µm² of the cortical tubulointerstitial (TI) area or of glomerular capillary tuft area. Cortical interstitial fibrosis was quantitated by PicroSirius red staining under polarized light by a computerized manner. Results: The uMCP-1 ratio (pg/ml/urinary creatinine mg/ml) was positively correlated (Spearman coefficient) with proteinuria (r = 0.4629; p < 0.005) and number of macrophages in the cortical TI area (r = 0.64; p = 0.0005), and negatively correlated with creatinine clearance (r = –0.4877; p < 0.001). The uMCP-1 ratio was not significantly correlated with number of macrophages/glomerular capillary tuft area (r = 0.27; p = 0.19) or with percent cortical interstitial fibrosis (r = 0.08; p = 0.62). Conclusions: The uMCP-1 excretion is a biomarker of the inflammatory activity of the TI area, and does not reflect chronic interstitial damage.

The NHE1 Na+/H+ exchanger regulates cell survival by activating and targeting ezrin to specific plasma membrane domains.

NHE1 is a ubiquitously expressed Na+/H+ exchanger, which is important for vital cell functions. Using in vivo models of kidney podocyte injury and renal tubular epithelial cell (RTC) culture systems, we previously demonstrated that NHE1 defends against apoptosis by a mechanism involving ezrin binding to the NHE1 cytoplasmic domain. We now extend the NHE1 role to diabetic mouse models and refine the mechanism of NHE1-dependent ezrin activation. Streptozotocin induced diabetes resulted in greater azotemia, albuminuria and tubulointerstitial pathology in NHE1-deficient swe/swe compared to wild-type control mice. Increased RTC apoptosis was noted in swe/swe mice, suggesting that loss of NHE1 function leads to tubular atrophy, which predicts kidney disease progression. In vitro, proximal RTC derived from swe/swe mice also underwent increased apoptosis in response to staurosporine or a hypertonic environment. Activated ezrin normally resides in the apical domain of the proximal RTC, while NHE1 is a basolateral protein. After NHE1 activation by intracellular acidification or extracellular hypertonicity, confocal immunofluorescence microscopy in polarized LLC-PK1 cells demonstrated transient ezrin localization to lateral membrane domains, where it is positioned to interact with NHE1. We conclude that cell stresses promote NHE1-ezrin interaction, which activate cell survival pathways to prevent apoptosis in diabetic and non-diabetic kidney diseases.

Acute sodium overload produces renal tubulointerstitial inflammation in normal rats

The aim of the present study was to determine whether acute sodium overload could trigger an inflammatory reaction in the tubulointerstitial (TI) compartment in normal rats. Four groups of Sprague-Dawley rats received increasing NaCl concentrations by intravenous infusion. Control (C): Na+ 0.15 M; G1: Na+ 0.5 M; G2: Na+ 1.0 M; and G3: Na+ 1.5 M. Creatinine clearance, mean arterial pressure (MAP), renal blood flow (RBF), and sodium fractional excretion were determined. Transforming growth factor beta1 (TGF-beta1), alpha-smooth muscle actin (alpha-SMA), RANTES, transcription factor nuclear factor-kappa B (NF-kappaB), and angiotensin II (ANG II) were evaluated in kidneys by immunohistochemistry. Animals with NaCl overload showed normal glomerular function without MAP and RBF modifications and exhibited a concentration-dependent natriuretic response. Plasmatic sodium increased in G2 (P < 0.01) and G3 (P < 0.001). Light microscopy did not show renal morphological damage. Immunohistochemistry revealed an increased number of ANG II-positive tubular cells in G2 and G3, and positive immunostaining for NF-kappaB only in G3 (P < 0.01). Increased staining of alpha-SMA in the interstitium (P < 0.01), TGF-beta1 in tubular cells (P < 0.01), and a significant percentage (P < 0.01) of positive immunostaining for RANTES in tubular epithelium and in glomerular and peritubular endothelium were detected in G3 > G2 > C group. These results suggest that an acute sodium overload is able 'per se' to initiate TI endothelial inflammatory reaction (glomerular and peritubular) and incipient fibrosis in normal rats, independently of hemodynamic modifications. Furthermore, these findings are consistent with the possibility that activation of NF-kappaB and local ANG II may be involved in the pathway of this inflammatory process.

Tubulointerstitial immune complex nephritis in a patient with systemic lupus erythematosus: role of peritubular capillaritis with immune complex deposits in the pathogenesis of the tubulointerstitial nephritis

Class IV-G (A/C) diffuse lupus nephritis and tubulointerstitial (TI) nephritis in a 31-year old woman was studied by light, immunofluorescence (IF), and electron microscopy (EM), to determine the pathogenesis of the TI lesions. The light microscopic findings showed peritubular capillaritis in the interstitium, with ruptures in the capillary structure, lysis of the surrounding tubular basement membrane (TBM), extravasated red blood cells (RBCs), the infiltration of neutrophils and mononuclear cells, and edema. The IF study revealed IgG, IgA, IgM, C1q, C3, and C4 depositions along the TBM, on the capillary walls, and in the interstitium proper. The EM study disclosed the deposition of immune complexes in the TBM, the capillary wall, and the interstitium proper. Based on these findings, the TI nephritis in this patient was considered to be due to peritubular capillaritis secondary to the immune complex depositions in the capillary wall of the interstitium.

Rituximab for Idiopathic Membranous Nephropathy

Rituximab effectively reduces proteinuria in patients with idiopathic membranous nephropathy (IMN), but response to treatment may vary from patient to patient. The association between baseline clinical, laboratory, and histology covariates and proteinuria reduction was evaluated retrospectively by multiple linear regression analysis at 3 mo after rituximab therapy in 14 patients with IMN with proteinuria > 3.5 g/24 h while on angiotensin-converting enzyme inhibition for at least 6 mo and no previous remissions. The association strength was expressed by standardized beta coefficients (SbetaC). Glomerular (SbetaC = 0.48, P = 0.049) and tubulointerstitial (TI) scores (SbetaC = 0.61, P = 0.003) predicted the outcome. Among glomerular and TI score components, tubular atrophy (SbetaC = 0.59, P = 0.003) and interstitial fibrosis (SbetaC = 0.60, P = 0.001) were significantly associated with 3-mo proteinuria. Urinary protein excretion decreased from 9.1 +/- 4.0 to 4.6 +/- 3.5 g/24 h (P < 0.001) in eight patients with TI score 1.7 but did not change in six with a score > or = 1.7. Nine additional patients with IMN then were allocated prospectively to rituximab treatment on the basis of a TI score < 1.7. Three-month proteinuria decreased in all patients from 8.9 +/- 5.3 to 4.9 +/- 3.9 g/24 h (P < 0.001) and serum albumin increased from 2.2 +/- 0.6 to 2.8 +/- 0.5 mg/dl (P < 0.01). Changes in serum albumin and cholesterol were inversely correlated (P < 0.02, r = -0.44). Rituximab achieved CD20 and CD19 depletion in all patients. In patients with IMN and nephrotic proteinuria despite angiotensin-converting enzyme inhibition therapy, renal biopsy findings may help in predicting response to rituximab and defining selection criteria for randomized trials that aim to assess the risk/benefit profile of B cell target therapy as compared with aspecific immunosuppressants and/or conservative therapy alone.

Renal Interstitial Fibrosis and Angiotensin Inhibition

Tubulointerstitial (TI) fibrosis is a final common pathway to progressive renal injury of all forms of renal disease. However, once renal damage reaches a certain threshold, progression of renal disease is consistent, irreversible, and largely independent of the initial injury. Angiotensin (AT) II is the main effector of the renin angiotensin system (RAS) and effects that may contribute to the onset and progression of renal damage. AT II may also directly contribute to accelerate renal damage by sustaining cell growth, inflammation, and fibrosis. Interventions that inhibit the activity of the RAS are renoprotective and may retard or even halt the progression of chronic nephropathies. Unilateral ureteral obstruction suggested as a well-established experimental model of progressive interstitial expansion and fibrosis. Although technically challenging, some investigators have successfully relieved the obstruction and reported significant reduction in interstitial fibrosis severity. Drugs that modulate the RAS, such as ACE inhibitors and angiotensin type 1 (AT1) receptor antagonists, have demonstrated protective renal effects and can ameliorate fibrosis. However, neither ACE inhibitor nor AT1 receptor blockade completely suppresses progression of renal disease. Dual blockade of the RAS with ACE inhibitors and AT1 receptor blockers may provide renal benefit beyond therapy with either drug alone, due to their potential additive beneficial effect.

Henoch–Schönlein purpura nephritis with nephrotic‐range proteinuria: histological regression possibly associated with cyclosporin A and steroid treatment

Objective: To clarify the therapeutic role of cyclosporin A (CyA) for patients with Henoch–Schönlein purpura nephritis (HSPN) showing nephrotic‐range proteinuria.Methods: The clinical and histological findings of eight children (7.7±3.8 years), who were treated with CyA and prednisolone, were evaluated retrospectively. All underwent a renal biopsy before therapy, and six of the eight patients received a follow‐up biopsy after therapy.Results: The histological grade of the International Study of Kidney Disease in Children (ISKDC) was improved in all six patients who received a follow‐up biopsy (pre‐therapy, four grade IIIa and two grade IIIb; post‐therapy, one grade I and five grade II) and it was statistically significant (p = 0.031). The activity index was significantly decreased after therapy (8.3±1.6 vs. 3.5±1.5, p = 0.031), and the chronicity index (0.5±0.5 vs. 0.7±1.0) and tubulointerstitial (TI) scores (1.5±1.3 vs. 0.8±1.6) did not change. There was a reduction in proteinuria from 3.2±2.3 to 0.1±0.1 g/m2/day (p = 0.008) and renal function remained normal in all patients after therapy. However, one patient showed CyA‐induced nephrotoxicity at a second biopsy. After an average follow‐up period of 3.8 years, six patients showed normal urine and renal function, and two showed minor urinary abnormalities.Conclusion: This study suggests that CyA therapy is effective in reducing proteinuria, which is a known risk factor for the development of renal insufficiency in HSPN and may regress the renal pathology in patients with nephrotic‐range proteinuria.

Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria.

Hyperoxaluria is a recognized cause of tubulointerstitial lesions and this circumstance could contribute to cause chronic renal disease. The renin-angiotensin system has a critical role in the development of interstitial fibrosis, mostly by angiotensin II type 1 receptor stimulation of pro-fibrotic mechanisms. We evaluated whether angiotensin II type 1 receptor blockade prevents oxalate renal lesions. We divided 2-month-old male Sprague-Dawley rats into 4 groups, namely group 1-control, group 2-hyperoxaluria, group 3-hyperoxaluria plus losartan and group 4-losartan. For 4 weeks groups 2 and 3 received 1% ethylene glycol (precursor for oxalates) in drinking water. Losartan (40 mg./kg. body weight) was administered in groups 3 and 4 daily. At the end of the study renal lesions were evaluated using anti-alpha-smooth muscle actin, anti-collagen type III, anti-monocytes/macrophages and anti-transforming growth factor-beta1 antibodies. To evaluate oxidative stress in renal tissue total glutathione and thiobarbituric acid reactive substances in kidney homogenates were determined. Regarding renal functional parameters, creatinine clearance and urinary albumin excretion were also studied. Despite similar urinary oxalate levels compared with group 2 group 3 rats showed fewer tubulointerstitial lesions, consisting of significant lower scores for tubular atrophy, unspecific inflammatory cell infiltrate, ED1 mouse anti-rat monoclonal antibody (Serotec, Ltd., Oxford, United Kingdom) (monocytes/macrophages), crystal deposits, interstitial fibrosis, alpha-smooth muscle actin, collagen type III and tubulointerstitial transforming growth factor-beta1. Moreover, urinary albumin excretion and creatinine clearance were significantly improved in group 3 (p <0.01). Higher total glutathione and lower thiobarbituric acid reactive substances were also observed in this group (p <0.01). Thiobarbituric acid reactive substances were the most important and significant independent variable correlating with interstitial fibrosis (t ratio 4.867, p <0.04). We believe that the renal-angiotensin system interaction by losartan produces a beneficial effect against renal lesions caused by hyperoxaluria through a number of actions, including a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors lead concurrently to preserve tubular epithelial cell and renal interstitium integrity. In addition, these results suggest that the principal mechanism of action should be mediated by angiotensin II type 1 receptors.

Vasodilator agents inhibit opossum kidney proximal tubular cell growth.

Renal tubule cell hyperplasia and hypertrophy have been regarded as antecedents of tubulointerstitial fibrosis. Dipyridamole and pentoxifylline inhibit rat glomerular mesangial cell growth. The purpose of this study was to examine the effects of pentoxifylline and other vasodilators on opossum kidney proximal tubular (OKP) cell growth. A modified 3-(4,5-dimethylthiazil-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate cell proliferation in OKP cells. Pentoxifylline caused a dose-dependent inhibition of serum-stimulated proliferation of OKP cells: inhibition was 17%, 25%, and 37% at 0.03 mg/mL, 0.1 mg/mL, and 0.3 mg/mL, respectively. Dipyridamole 1.7 mg/L and 6 mg/L resulted in 34% and 46% inhibition, respectively. Verapamil 10(-6) M, 10(-5) M, and 10(-4) M resulted in 34%, 46%, and 67% inhibition, respectively. These inhibitory effects may contribute to renal protection or retardation of renal disease progression.

Alterations in Gene Expression in Cadaveric vs. Live Donor Kidneys Suggest Impaired Tubular Counterbalance of Oxidative Stress at Implantation

Recipients of live donor transplant kidneys (LIV) exhibit a significantly longer allograft half-life compared with cadaveric donor organs (CADs). The reasons are incompletely understood. Therefore this study sought to elucidate the genome-wide gene expression profiles in microdissected transplant kidney biopsies obtained from five cadaveric and five matched live donors before transplantation. cDNA microarrays were used to determine the transcripts in isolated glomeruli (G) and the tubulointerstitial (TI) compartment. Data were subjected to hierarchical clustering, maxT adjustment and a jackknife procedure to ensure robustness of reported findings; validation was performed by independent analysis of split biopsies and TaqMan-PCR.One hundred and thirteen sequences representing 62 unique genes (17 redundant features), and 34 ESTs separated G from TI. No difference in gene expression was found in G between LIV and CAD kidneys, but nine genes (two represented twice) and three ESTs were abundantly expressed in the CAD TI compared with LIV. The main biological function of these genes is counter regulation of oxidative stress. Promoter analysis of significant features suggested coregulated gene groups.These data suggest that CAD kidneys exhibit a distinctly different set of transcripts in the TI compartment but not in the G compartment when compared with LIV kidneys.

Tubulointerstitial nephritis and uveitis syndrome (TINU syndrome) with unilateral neuroretinitis: a case report.

To report a case of tubulointerstitial nephritis and uveitis syndrome (TINU syndrome) with unusual unilateral neuroretinitis. An 11-year-old girl who developed anterior uveitis 3 months after acute tubulointerstitial nephritis, without any etiologic factors for either nephritis or uveitis, is presented. Several days after the anterior uveitis, the patient presented with unilateral optic disk edema associated with a serous retinal detachment of the posterior pole, never previously described in TINU syndrome, leading to the formation of perifoveal intraretinal exudates. In spite of these posterior lesions, clinical course confirmed the previously reported favorable visual outcome of TINU syndrome treated with corticosteroids. It appeared that ocular impairment in TINU syndrome could be more diffuse and more extensive than previously described. As well as the whole uveal tract, the retina and the optic nerve may also be the ocular targets of the idiopathic inflammatory response in TINU syndrome. (Eur J Ophthalmol 2004; 14: 334-7).

Reversal of glomerulosclerosis after high-dose enalapril treatment in subtotally nephrectomized rats.

Interventions to block the renin-angiotensin system (RAS) halt the progression of renal lesions in renal damage models. It has recently also been reported that established glomerulosclerosis can be reversed by pharmacologic blockade of the RAS. It was the aim of this study to confirm that high doses of angiotensin-converting enzyme (ACE) inhibitors reverse established glomerulosclerosis and to extend the findings by providing quantitative information on glomerular geometry, podocytes and other glomerular cells, renal vessels and tubulointerstitial tissue. Male Sprague Dawley rats were subjected to subtotal surgical renal ablation (SNX) (n = 27) or sham operation (n = 31) and fed using a pair-feeding protocol. Eight weeks after surgery, rats were either sacrificed or allocated to two arms: enalapril treatment (48 mg/kg body wt per day administered in the drinking fluid for 4 wk) or no treatment. Renal morphology was evaluated after 8 or 12 wk, respectively, by stereology in tissue fixed by pressure-controlled perfusion. Both systolic BP and albumin excretion rate were significantly higher in SNX compared with sham-operated controls. They were significantly reduced in SNX after delayed enalapril treatment. The glomerulosclerosis (GSI), tubulointerstitial (TII), and vascular (VI) damage indices were significantly higher in all SNX groups than in sham-operated controls. At the end of the experiment (12 wk after SNX) GSI, TII, and VI were significantly lower in SNX with delayed enalapril treatment (0.77 +/- 0.18, 0.63 +/- 0.19 and 0.43 +/- 0.16, respectively) compared with untreated SNX (1.64 +/- 0.14, 1.16 +/- 0.34 and 0.67 +/- 0.29, respectively). GSI, TII, and VI were also significantly lower in SNX with delayed enalapril treatment compared with SNX sacrificed without treatment 8 wk after SNX. The same was true for glomerular volume. The number of podocytes was not affected by SNX, but podocyte volume was increased. Both indices remained unaffected by treatment. The numbers of cells within the mesangium and endothelial cells per glomerulus were significantly lower in SNX after delayed enalapril treatment compared with untreated SNX. These results strongly suggest regression of preexisting lesions, i.e., glomerular, tubular, and vascular remodeling as well as reversal of glomerular hypertrophy by ACE inhibitor treatment. The study confirms that high-dose ACE inhibitor treatment causes partial reversal of glomerular as well as interstitial lesions in subtotally nephrectomized rats.

Glomerular hemodynamic changes associated with arteriolar lesions and tubulointerstitial inflammation

Glomerular hemodynamic adaptations to loss of renal mass are thought to be the initiating factor of progression to renal failure; however, tubulointerstitial (TI) injury correlates better with progression than with glomerular damage. Thus, it is conceivable that tubulointerstitial alterations participate in the pathophysiology of renal disease progression by modifying the adaptive responses of glomerular hemodynamics. In experimental models of progressive renal disease, suppressing tubulointerstitial inflammatory cell infiltration with anti-inflammatory drugs reduces renal damage despite persistence of systemic hypertension. In recent studies in rats with subtotal renal ablation, we found that treatment with polysulphate pentosan (PPS) and with mycophenolate mofetil (MMF) prevented proteinuria, glomerular hypertension, and hyperfiltration, despite persisting arterial hypertension due to higher afferent resistance. In addition, arteriolopathy was significantly attenuated by MMF, suggesting preservation of vascular structure and function. Association of vascular injury of afferent arterioles, glomerular hemodynamic changes, and renal lesions has been described in other conditions such as hyperuricemia, protein overload, fawn-hooded rats, and aging spontaneously hypertensive rats (SHR). Arteriolopathy results in a maladaptive function that permits the transmission of systemic hypertension to glomerular capillaries. Glomerular hypertension results in mechanical damage to the capillary wall and increased filtration of proteins to tubular lumen. Enhanced tubular reabsorption induces synthesis of proinflammatory and profibrotic factors, resulting in tubulointerstitial inflammation and fibrosis. In conditions in which there is overactivity of the renin-angiotensin system (RAS), such as mild hyperuricemia and protein overload, arteriolopathy is associated with increased glomerular pressure and reduced glomerular plasma flow that results in post-glomerular ischemia and tubulointerstitial injury.

Exploring the connection between chronic renal fibrosis and bone morphogenic protein-7.

Tubulointerstitial fibrosis is a hallmark feature of chronic renal injury. Specific therapies to control the progression of renal fibrosis towards end-stage renal failure are still limited. Transforming growth factor-beta1 (TGF-beta1) has been identified as a major mediator of renal fibrosis. Recent reports have suggested that Bone Morphogenic Protein-7 (BMP-7), another member of the TGF-beta superfamily, accelerates repair of acute renal injury and ameliorates progression of chronic renal fibrosis in a variety of animal models. Interestingly, BMP-7, an endogenous molecule which is present in the normal kidney, vastly decreases its expression during renal injury. Although, the mechanism of BMP-7 action in the kidney is not yet fully understood, the idea of an endogenous molecule with reno-protective function is intriguing.

Evaluation of the rate of progression of chronic renal transplant rejection using the proliferating cell nuclear antigen

Since the introduction of kidney transplantation, the short-term patient and graft survival have been progressively improving, but the long-term graft survival and half-life of transplants have not. Beyond doubt, chronic rejection (CR) remains the major cause of chronic graft failure and is responsible for the loss over 10 years of 50% of grafts. (1-4). CR is characterized clinically by proteinuria, hypertension and declining renal function, and pathologically by arteriosclerosis, glomerulosclerosis and interstitial fibrosis. The progressive morphological changes of glomerular, vascular and tubulointerstitial (TIN) compartments in renal allografts experiencing chronic rejection correlate directly with declining renal function and the eventual graft loss [5]. In consideration of this fact and using proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation, a retrospective analysis of renal allograft biopsies was carried out with the aim to determine whether quantification of the cell proliferation activity in three tissue compartments may be used as a prognostic index of CR progression. Clinical course and biopsy specimens of 27 patients with clinically and morphologically confirmed (Banff schema) diagnosis of CR were retrospectively evaluated. Patients were transplanted between 1988 and 1994 at our Institute and regularly followed-up for at least 2 years after transplantation (14 patients), or till haemodialysis has been resumed (11 patients) or till death (2 patients). The progression rate of renal graft function deterioration was estimated by slopes of regression lines obtained by plotting 1/sCr vs time. According to the progression rate after biopsy patients were divided into two groups: fast progression group (10 patients) and slow progression group (17 patients). Immunosuppressive regimens included induction with anti-lymphocyte globulin followed with cyclosporine, steroids and azathioprine. Immunohistochemistry. Sections of formalin-fixed, paraffinembedded tissue were stained for PCNA with monoclonal mouse anti-proliferating cell nuclear antigen (clone PC 10, Dako) as follows in ref. [6]. PCNA immunoreactivity of smooth muscle cells and lymphocytes in vascular intima, lymphocytes in TIN and glomerular mesangium proliferation was assessed by semiquantitative scoring (scale 0-3). Normal tonsil was used as a positive control. Differences between groups were evaluated using Student's t-test. Pearson's correlation test was used to study the relationship between variables. P values < 0.05 were considered as significant. Data on patients are presented in Table 1. The only statistical difference was in progression of chronic graft failure presented by regression lines obtained by plotting 1/sCr vs time. PCNA protein expressions in vascular, glomerular and TIN compartments for both groups are presented in Figs. 1-3.. PC 10 immunoreactivity was confined to the nucleus. The pattern of staining was granular or diffuse. Individual values of the scores of PCNA immunoreactivity in different tissue compartments for the groups examined are presented in Tables 2 and 3. Comparison of the means of PCNA immunoreactivity scores in different tissue compartments of fast and slow progression group showed that cells proliferation in glomerular, TIN and vascular compartments are significantly higher in the fast than in the slow progression group (Table 4). Besides, significant positive correlation was found between the slopes of the regression lines plotting 1/sCr vs. time and cells proliferation scores for all compartments (Table 5). Chronic graft failure may occur due to CR, cyclosporine nephrotoxicity, repeated acute rejection, recurrent glomerular diseases, surgical and urological complications, but CR was recognized as the most common cause of chronic graft failure and renal graft loss [7]. Progression of renal disease reflects the interactive effects of changes in the structure and function. This notion has been examined many times in the native kidneys by studies correlating glomerular filtration rate with architectural deterioration in the glomerular or interstitial compartment [8-10]. Meanwhile, the similar studies in human renal allograft are scarce. Increased PCNA immunodetection in glomerular cells as well as in interstitial infiltrates was described in acute and chronic renal graft rejection and diagnostic value of this finding was analyzed [12, 15]. Similarly, PCNA was often examined immunohistochemically in lymphoma and solid tumours as a marker of cell proliferation, but its reliability as a prognostic factor was also evaluated [18-20]. In the present study the prognostic value of PCNA expression in different tissue compartments of renal grafts experiencing CR was examined for the first time. The present study revealed that two groups examined with the different chronic graft failure progression rate as the main difference had significantly different proliferation of cells in glomerular, TIN and vascular compartments. PCNA immunoreactivity scores in these three compartments were significantly higher in the fast than in slow progression group. The significant positive correlation between the slope of the regression line plotting 1/sCr vs. time and cell proliferation in glomerular, TIN and vascular compartments was also found. Therefore, it was suggested that the measurement of PCNA expression in renal graft tissue might be used as a prognostic index.

Evaluation of P4504a Genes for Hypertension and Renal Disease Using Chromosome 5 Congenic Strains of Dahl S Rats.

53 P4504A1 and 4A2 genes are differentially expressed in the kidney and cosegregate with blood pressure in an F 2 cross of Dahl S and Lewis rats; however, it is unclear whether this pathway plays a causal or secondary role in the development of hypertension. The present study examined whether transfer of overlapping segments of chromosome 5 from D5Rat84 to D5Rat34 that excludes (4A- strain) or includes all four P4504A genes(4A+ strain),or just the 4A3 and 4A8 genes (4A3,8+ strain), of a Lewis rat alters blood pressure, proteinuria, and renal injury in 12 week old congenic strains of Dahl S rats fed a high salt diet (8% NaCl) for 3 weeks. MAP measured in conscious rats using a chronic catheter averaged 188 ± 3 mm Hg in male Dahl S rats (n=24), 190 ± 3 mm Hg in the 4A- congenic strain (n=24), 169 ± 4 mm Hg in the 4A+ congenic strain (n=30), and 172 ± 3 mm Hg in the 4A3,8+ strain (n=33). Transfer of the P4504A region also significantly reduced proteinuria, renal hypertrophy, and the degree of glomerular and tubulointerstitial injury. Protein excretion averaged 246 ± 22 mg/day in male Dahl S rats and 255 ± 25 mg/day, 129 ± 8 mg/day, and 169 ± 12 mg/day in the 4A-, 4A+, and 4A3,8+ strains, respectively. The antihypertensive and renoprotective effects of transfer of the P4504A region were associated with signficant increases in the expression of P4504A mRNA, P4504A protein levels, and the renal formation of 20-HETE. Interestingly, transfer of the P4504A region had no effect on blood pressure in female rats of the 4A+ or 4A3,8+ congenic strains, but it still reduced the degree of proteinuria and renal injury. These studies exclude the P4504A1 and 4A2 loci as the causal genes in this region in the development of hypertension in Dahl S rats. However, they indicate that the P4504A3 and 4A8 genes still lie in the interval between D5Rat108 and D5Rat54 that contains a locus that reduces blood pressure, is renoprotective, and alters the expression of P4504A genes in Dahl S rats.

Enalapril (E) Attenuates Oxidative Stress (OXS) in Diabetic Rats.

P183 Oxidative stress has been involved as a possible mechanism of tissue injury. The effect of E on OxS and tissue injury was studied in kidney and heart from streptozotocin (STZ)-induced diabetic rats. Male Sprague-Dawley, were divided into: STZ (65 mg/kg, single i.p. dose); STZ+E (STZ and E, 20 mg E /L drinking water), and Control (C) (saline i.p. and tap water). At 6 mo. organs were studied by light microscopy. Glomerular (GML) and tubulointerstitial lesions (TIL) were graded by a semiquantitative score (0-4). Glycemia, creatinine clearance (CrCl) and proteinuria were determined. OxS was evaluated by determining total glutathione content (GSH), protein-associated sulfhydryls (SH) and 2-thiobarbituric reactive substances (TBARS, an indicator of lipid oxidation) in kidney and heart homogenates. In STZ rats E decreased proteinuria, GML and TIL,and increased CrCl. In kidney and heart, E attenuated OxS associated to STZ. Results suggest that, in addition to previously described mechanisms, E might protect renal and cardiac tissue by attenuating OxS in STZ-induced diabetic rats.